(1) Field of the Invention
The present invention relates to an alkaloid compound that inhibits biosynthesis of particular products of secondary metabolism. In particular, the present invention relates to an alkenylene piperidine amide wherein the alkenylene is a C18 alkenylene with one or more double bonds, which can be isolated from Piper nigrum, that inhibits transcription of fungus genes nor-1, tri5, ver-1, verA, fas-1a, omt-1, alfR and ipnA. The present invention further relates to a method for identifying compounds that inhibit the biosynthesis of mycotoxins in fungi. In particular, a method for identifying compounds that inhibit biosynthesis of aflatoxin in Aspergillus spp. and deoxynivalenol in Gibberella spp.
(2) Description of Related Art
Mycotoxins are a group of structurally heterogeneous secondary metabolites produced by a diverse group of fungal plants pathogens. Infestation of crops and food commodities by mycotoxin producing fungi is a serious problem in view of the immunosuppressive, carcinogenic, cytotoxic, and teratogenic effects of the compounds in humans and animals. One of the most economically important mycotoxins worldwide is aflatoxin, a polyketide produced by several Aspergillus spp. Aflatoxin is the best studied of the mycotoxins and much of the molecular biology of the biosynthetic pathway has been determined in Aspergillus flavus, Aspergillus parasiticus, and Aspergillus nidulans. Aspergillus flavus produces aflatoxin B1 and aflatoxin B2 whereas Aspergillus parasiticus produces in addition aflatoxin G1 and G2. Aspergillus nidulans, which is not considered to be an agricultural threat, has been used as a model genetic system for studies of aflatoxin biosynthesis because it produces sterigmatocystin, an aflatoxin precursor. The genes for aflatoxin biosynthesis are clustered in all three species. The molecular biology of aflatoxin biosynthesis is reviewed by Trail et al., in Microbiol. 141: 755–765 (1995). Aspergillus flavus and Aspergillus parasiticus are weak pathogens of corn, cotton, peanut, and nut crops: their effect is limited to a slight reduction in crop yield. However, the significant consequence of crops infected with either of these fungi is contamination by aflatoxin, which is produced under certain conditions during the infection. Traditional control strategies such as breeding crops for resistance to the fungi or chemical treatments of crops to prevent infection by the fungi have not been effective.
Aflatoxin is a secondary metabolite that appears to be the most potent naturally occurring carcinogen known (Council for Agricultural Science and technology (CAST), 1989). It is suspected of being responsible for the high incidence of human liver cancer in many areas of the world (Eaton and Gallagher, Ann. Rev. Pharmacol. Toxicol. 34: 135–139 (1994)). Aflatoxin is introduced into the food chain by preharvest and postharvest contamination of foods and feeds. Also, products from animals that have been fed aflatoxin contaminated feed may also become contaminated. Currently, the U.S. Food and Drug Administration limits the allowable amount of aflatoxin in food to 20 ppb, with slightly higher levels allowed in feeds. Because the level of aflatoxin in products destined for human consumption is strictly regulated in the U.S., aflatoxin contamination is primarily of economic importance. However, even though aflatoxin levels in foods is limited to 20 ppb, the effect of chronic exposure to low levels of aflatoxin on human health is unknown. Thus, some European countries require the presence of aflatoxin in foods intended for human consumption to be 0 ppb. In areas of the world where regulations do not exist, aflatoxin is a serious health problem (CAST, 1989).
Approaches to control of aflatoxin have been broadly grouped into preharvest and postharvest strategies. Proper grain storage can greatly reduce contamination postharvest, and some decontamination methods, while costly, are used, e.g., ammoniation. However, most research efforts at control of aflatoxin has been directed at the preharvest elimination of infection and contamination, since the ability to control preharvest contamination would reduce the need for postharvest elimination. Preharvest methods have included agricultural practices such as irrigation strategies designed to eliminate stress to crops associated with drought, which appears to increase production of aflatoxin by the fungus. Other methods include using regionally adapted varieties of crop plants. However, these methods have been expensive to implement and have not been completely effective. Chemical control methods have also been ineffective at controlling infection by these fungi.
The development of host plants that are resistant to Aspergillus infection and aflatoxin contamination has not been as successful as have programs for breeding resistance to other pathogens. In general, the resistant varieties that have been made are unstable from growing season to growing season and from region to region. Also, screening plants for resistance to colonization by Aspergillus spp. and aflatoxin contamination has been difficult. In corn, and frequently in cotton, inoculation methods have been difficult, often requiring wounding the plant to introduce the fungus, which may overwhelm the plants natural resistance reactions making it difficult to evaluate the plants resistance mechanisms (Cotty, Plant Dis. 73: 489–492 (1989)).
Methods have been developed for inhibiting mycotoxin production in crops. For example, U.S. Pat. No. 5,942,661 to Keller discloses a method of inhibiting mycotoxin production by introducing into the plant a gene encoding a lipoxygenase pathway enzyme of the mycotoxin. The method may produce transgenic plants that are substantially resistant to mycotoxin contamination. Mycotoxin resistance is further increased by introducing into the plant antisense genes for the 9-hyperoxide fatty acid producing lipoxygenases. However, reducing aflatoxin contamination by making transgenic plants resistant to aflatoxin production is expensive and time consuming, and since transformation efficiencies varies from plant species to plant species, the method may not be successful for all plant species. Furthermore, the long-term effect of introducing transgenic plants into the environment is unknown.
Since traditional methods for controlling fungal infection and/or production of aflatoxin by breeding, chemicals, or transgenic plants have not been completely effective, there is a need for an inexpensive and effective method for either controlling infection of crops by fungi such as Aspergillus spp. or Gibberella spp., or controlling the biosynthesis and accumulation of mycotoxins such as aflatoxin or deoxynivalenol in plants infected with fungi such as Aspergillus spp or Gibberella spp., respectively. There is also a need for a rapid and inexpensive method for identification of chemicals or compounds in natural extracts that inhibit production of mycotoxins such as aflatoxin and deoxynivalenol.